Material transfer is required in various situations. In particular, substance transfer techniques often become a significant challenge when mechanically reproducing manual operations. For example, biological or chemical experiments are made up of basic operations, such as mixing, reacting, and separating substances. Automation of such operations require a combination of various substance transfer techniques.
In the field of analytical chemistry, microchips have been receiving attention. A microchip is a device wherein substances can be reacted and separated in flow channels formed on glass or plastic surfaces. Microchannels with widths and depths in the range of several micrometers to several hundred micrometers are formed on the microchip. These microchannels are used as cavities for reaction and separation. The use of such cavities, which have minute internal volumes, is a characteristic microchip feature.
Microchannels not only have a reduced internal volume, but also enable transfer of liquids by electroosmosis. Electroosmosis is a phenomenon that takes place on a glass surface or the like which comprises silanol groups (—SiOH), whereby a solvent to which voltage is applied moves toward the cathode. The solvent flow caused by electroosmosis is called electroosmotic flow. On a microchip, solvent can be transported using electroosmotic flow as the driving force, without using a pump or other means of transport.
Due to the above characteristics, microchips are expected to have the following advantages:
The device size itself is small;
The amount of sample required for analysis or separation is small;
Since the amount of sample is small, the period required for reaction, separation, and analysis is short;
The device can be produced inexpensively using a replica; and
Solvent transport is possible independent of mechanical means, such as pumps or valves.
There are many reports of using these advantages in the application of microchips to protein or nucleic acid separation, analysis, and reaction. One such microchip already in practical use is a microchip for electrophoresis.
For example, the Microchip Electrophoresis System “MCE-2010” (Shimadzu Corporation) uses a microchip comprising channels with widths and depths of 10 μm to 100 μm, formed on quartz glass. This microchip is equipped with electrodes necessary for electrophoresis, and also with a hole for supplying electrophoretic buffer, and a slit for optical detection. The channels formed on the microchip function as cavities for electrophoretic separation, as well as channels for supplying sample and electrophoretic buffer from the exterior of the device.
Also known is a microchip for electrophoresis, which uses a number of channels, concentrically located on a circular substrate (Non-patent document 1: Paegel B. M. et al., Proc. of μTAS2001: 462-464, 2001). Since a circular substrate is used, all channels can be optically scanned by rotating the substrate.
In these known microchips, electrophoretic separation is conducted within one continuous channel. Such a structure is not problematic, as long as the electrophoretic separation uses a single medium. However, it is difficult to use this same microchip structure in the continuous electrophoretic separation of combinations of different media.
Generally, physical barriers such as valves or walls for controlling liquid flow cannot be provided in the channels of a microchip. Accordingly, a number of intersecting channels have been used on microchips in an attempt to control the timing of contacting different liquids (Non-patent document 2: Roy D. et al., Anal. Chem. 2000 72, p 5244-5249). When the channels intersect, the timing of contact of different liquids can be controlled by controlling the flow of liquids approaching an intersection. However, such microchips have only one channel for each of the different solvents. As a result, there is a risk that introduction of the second dimensional electrophoretic medium would mix the substances separated by the first dimensional electrophoresis.
Electrophoretic analysis is one analytical technique whose application to microchips has been attempted from an early stage. As mentioned above, microchips for use in electrophoresis are already commercially available. The present inventors have also filed a patent application for an analysis method using two-dimensional electrophoresis, which can be applied to microchips (WO 00/52458). Two-dimensional electrophoresis is important as an analytical technique that provides information extremely important to protein profiling.
In two-dimensional electrophoresis, electrophoresis must be carried out twice, using two different electrophoretic media. For example, two-dimensional electrophoresis using the following combination is common in protein profiling:
First: Isoelectric focusing by capillary gel electrophoresis
Second: Denatured gel electrophoresis using a slab gel
Typically, the gel in the capillaries is collected after completion of the first dimensional electrophoresis, and contacted with the slab gel before starting the second dimensional electrophoresis. However, such a procedure is unrealistic on a microchip. Accordingly, a structure was employed comprising a number of cavities branching from the cavity accommodating the first dimensional electrophoretic medium, where the second dimensional electrophoretic medium was placed in these cavities (Patent document 1: WO 00/52458). In this structure, the first and second dimensional electrophoretic media are always in contact during first dimensional electrophoresis. Since an electric drive force is only generated in the first dimensional electrophoretic medium, diffusion of test substances to the second dimensional electrophoretic medium, placed in a branched cavity during the first dimensional electrophoresis, was not deemed a serious problem.
However, in fact there is some possibility that contact of the media for different electrophoreses could vary the electrophoreses conditions. For example, in isoelectric focusing media, the pH gradient has a considerable effect on the electrophoretic analysis results. Accordingly, if the pH of isoelectric focusing media is changed by contact with the second dimensional electrophoretic medium, there is some risk that the isoelectric focusing conditions will change. In other words, during first dimensional electrophoresis, the medium is preferably isolated from the second dimensional electrophoretic medium. However, controlling the timing of media separation and contact is difficult using known microchip structures.
Two-dimensional electrophoresis on a microchip using the principle of capillary electrophoresis has also been attempted (Non-patent document 3: Anal. Chem. 2002, 74, 1772-1778). In this report, however, the top and bottom surfaces of the chip were peeled off after completion of the first dimensional electrophoresis, and the substrate materials for the second dimension were freshly attached to the top and bottom surfaces. In other words, the document does not disclose any specific means for controlling the timing of media separation or contact.